Pulmonary endothelial cells are a rich source of nitric oxide (NO) which is generated from L-arginine by the endothelial isoform of NO synthase (eNOS). NO production by endothelial cells can be increased by extracellular L-arginine despite a saturating a intracellular arginine concentration for eNOS. This observation has been termed the "arginine paradox" and can not be explained based on available data. We hypothesize that a caveolar complex between eNOS and the cationic amino acid transporter (CAT-1) responsible for arginine transport exists in lung endothelial cells. Such a complex provides an efficient mechanism for the directed delivery of substrate to eNOS and would account for the "arginine paradox". Since caveolae interact with the cytoskeleton through actin- associated proteins and microtubules, we also hypothesize that L-arginine transport is regulated by membrane-cytoskeleton interactions. Finally, based on work from the PI's laboratory, we hypothesize that hypoxia inhibits L-arginine transport by disrupting membrane-cytoskeleton interactions through a calpain-mediated mechanism. To verify that the CAT- 1 transporter is localized to caveolae in lung endothelial cells, we will use immunohistochemistry and deconvolution fluorescence microscopy to localize CAT-1, and we will isolate caveolae and identify the presence of the CAT-1 transporter by immunoblot analysis and transport assays. We will also use immunohistochemistry and deconvolution microscopy to determine whether CAT-1 and eNOS co-localize caveolae. We will confirm the existence of a caveolar complex by immunodepletion assays and fractionation of CAT- 1-eNOS protein complexes on sucrose gradients. To define membrane cytoskeletal interactions that affect CAT-1-mediated L-arginine transport, we will selectively disrupt the actin-microfilament and microtubule systems in lung endothelial cells and then measure CAT-1 mediated transport. We will identify qualitative and quantitative changes in cytoskeletal elements responsible for alterations in arginine transport. Finally, we will use immunohistochemistry, deconvolution fluorescence microscopy, and immunoblot analyses to assess the effect of hypoxia on the cytoskeleton and arginine transport with special emphasis on the role of calpain in mediating the hypoxic effects. These studies will advance our understanding of the mechanisms that regulate arginine transport and NO production in lung endothelial cells and will ultimately lead to new and improve ways to attenuate or prevent pulmonary vascular dysfunction.